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Article A Novel Truncating in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family

María Lachgar 1,2,† , Matías Morín 1,2,† , Manuela Villamar 1,2, Ignacio del Castillo 1,2 and Miguel Ángel Moreno-Pelayo 1,2,*

1 Servicio de Genética, Hospital Universitario Ramón y Cajal, IRYCIS, Carretera de Colmenar km 9.100, 28034 Madrid, Spain; [email protected] (M.L.); [email protected] (M.M.); [email protected] (M.V.); [email protected] (I.d.C.) 2 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28034 Madrid, Spain * Correspondence: [email protected] † These authors have contributed equally to this work.

Abstract: Nonsyndromic hereditary hearing loss is a common sensory defect in humans that is clinically and genetically highly heterogeneous. So far, 122 genes have been associated with this disorder and 50 of them have been linked to autosomal dominant (DFNA) forms like DFNA68, a rare subtype of hearing impairment caused by disruption of a stereociliary scaffolding (HOMER2) that is essential for normal hearing in humans and mice. In this study, we report a novel HOMER2 variant (c.832_836delCCTCA) identified in a Spanish family by using a custom NGS targeted panel (OTO-NGS-v2). This frameshift mutation produces a premature stop   codon that may lead in the absence of NMD to a shorter variant (p.Pro278Alafs*10) that truncates HOMER2 at the CDC42 binding domain (CBD) of the coiled-coil structure, a region that is essential Citation: Lachgar, M.; Morín, M.; for protein multimerization and HOMER2-CDC42 interaction. c.832_836delCCTCA mutation is Villamar, M.; del Castillo, I.; placed close to the previously identified c.840_840dup mutation found in a Chinese family that Moreno-Pelayo, M.Á. A Novel truncates the protein (p.Met281Hisfs*9) at the CBD. Functional assessment of the Chinese mutant Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive revealed decreased protein stability, reduced ability to multimerize, and altered distribution pattern DFNA68 Hearing Loss in a Spanish in transfected cells when compared with wild-type HOMER2. Interestingly, the Spanish and Chinese Family. Genes 2021, 12, 411. https:// frameshift might exert a similar effect at the protein level, leading to truncated mutants doi.org/10.3390/genes12030411 with the same Ct aberrant protein tail, thus suggesting that they can share a common mechanism of pathogenesis. Indeed, age-matched patients in both families display quite similar hearing loss Academic Editors: Santiago phenotypes consisting of early-onset, moderate-to-profound progressive hearing loss. In summary, Rodriguez and Laura Crisponi we have identified the third variant in HOMER2, which is the first one identified in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations, Received: 20 December 2020 and also to clarifying the genotype–phenotype correlations of DFNA68 hearing loss. Accepted: 9 March 2021 Published: 12 March 2021 Keywords: hereditary hearing loss; next-generation sequencing; custom panel; HOMER2; CDC42

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Hearing loss is the most common sensory deficit in humans, affecting around 1 in 1000 newborns. Its prevalence increases with the age up to 6–8% in the adult population, having a strong impact on the individual’s social isolation [1]. Genetic causes account for 50–60% of newborn hearing loss, 30% of which are nonsyndromic forms of deafness [2]. The genetic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. etiology of hearing impairment is highly heterogeneous. Up to date, 160 nonsyndromic This article is an open access article sensorineural hearing loss (NSSNHL) loci have been mapped, of which 122 genes have been distributed under the terms and identified [3]. Sixty-seven of these loci and 50 of these genes are associated with autosomal conditions of the Creative Commons dominant NSSNHL, being, in most cases, rare forms of post-lingual and progressive Attribution (CC BY) license (https:// hereditary hearing impairment. creativecommons.org/licenses/by/ Autosomal dominant NSSNHL-linked genes encode with a wide variety of 4.0/). functions [4], such as proteins (ACTG1, DIAPH1, PLS1), adhesion proteins (GJB2,

Genes 2021, 12, 411. https://doi.org/10.3390/genes12030411 https://www.mdpi.com/journal/genes Genes 2021, 12, 411 2 of 9

GJB3, GJB6, TJP2), motor proteins (myosins), scaffolding proteins (HOMER2), extracellular matrix proteins (TECTA, COL11A2), proteins involved in ion homeostasis, such as ionic channels (KCNQ4), transcription factors (EYA4, POU4F3), and even a microRNA (MIR96)[5]. The HOMER2 gene maps to 15q24.3 [6] within the DFNA68 critical interval and consists of nine exons. Two human transcript variants have been described (NM_004839.4 and NM_199330.3) as encoding the short isoform 1 (NP_004830.2, 343aa) and the long isoform 2 (NP_955362.1, 354aa) of HOMER2, respectively. HOMER2 belongs to a protein family encompassing three members: HOMER1 (MIM604798), HOMER2/CUPIDIN (MIM604799), and HOMER3 (MIM604800). Like HOMER2, members 1 and 3 of the family have long and short isoforms due to [7]. HOMER family members are scaffolding proteins that play a key role in Ca2+ signaling [8–10], mostly at the Post- Synaptic-Densities (PSD) [11], where they interact with G-protein coupled metabotropic glutamate receptors (mGluRs) [12] and regulate excitatory signal transduction and re- ceptor plasticity [7]. In their structure, HOMER proteins present a conserved N-terminal domain known as Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) 1 (EVH1) domain [7,13] that binds to proline-rich sequences (i.e. Pro-Pro-x-x-Phe, Pro-x-x- Phe or Leu-Pro-Ser-Ser-Pro, where x represents any amino acid) and a C-terminal domain that consists of a coiled-coil (CC) structure that includes a CDC42 binding domain (CBD) and two Leucine Zipper (LZA and LZB) motifs [7,14]. This C-terminal fragment mediates self-association with other Homer family members [7] and the interaction with the small GTPase CDC42 [15] through its CBD. In mice, Homer2 is widespread in the developing and maturing brain [16]. Recently, a study has shown that this gene is also expressed in a wide variety of developing tissues, including tooth, eye, cochlea, salivary glands, olfactory and respiratory mucosae, bone, and taste buds, being highly concentrated at puncta [17]. HOMER2 exhibits overlapping distribution patterns with HOMER1 and HOMER3, although they are distributed at distinct subcellular domains in several cell types [17,18]. Within the inner ear, HOMER2 is expressed in the stria vascularis, the Reissner’s membrane, and the inner and outer hair cells of the organ of Corti, especially in the stereocilia but also in perinuclear puncta and the cytoplasm [17,18]. Mild expression is observed in the vascular endothelium of the cochlea and the spiral ganglion [12]. Mice homozygous for the targeted deletion of Homer2 display early-onset rapidly progressive hearing loss [18]. HOMER2 was firstly associated with hearing loss by Azaiez et al. [18] who identified a heterozygous missense variant (p.Arg196Pro) in a European ascent family. A second mutation in HOMER2 (c.840_840dup; p.Met281Hisfs*9) was identified by Lu et al. [19] by Whole Exome Sequencing (WES) in a Chinese family segregating with hearing loss. Here, we present a third variant in HOMER2 (c.832_836delCCTCA, p.Pro278Alafs*10, NM_199330.3). This variant represents the second truncating mutation that affects the CBD and lead, as in the previous two families, to post-lingual and progressive hearing loss. This mutation is the first one identified in the Spanish population, thus increasing the mutational spectrum of this gene associated with DFNA68, a rare form of autosomal dominant nonsyndromic progressive hearing loss.

2. Patients and Methods 2.1. Patients Selection Patients and healthy relatives of family S1074 (Figure 1A) were recruited from the Uni- versity Hospital Ramón y Cajal (Madrid-Spain). Clinical history ruled out environmental factors as the cause of the hearing loss in the probands, and physical examination did not reveal any evidence of syndromic features. No other clinically significant manifestations, including balance or visual problems, were reported by any of the affected individuals. The hearing level was evaluated through pure tone audiometry. Air conduction thresholds were determined at frequencies ranging from 250 to 8000 Hz according to standard protocols. This study was designed in compliance with the tenets of the Helsinki Declaration, and patient enrolment was approved by the ethics committee and the human research Institu- Genes 2021, 12, 411 3 of 9

tional Review Boards of Hospital Ramón y Cajal (IRB number: 288-17). All participants of the family approved of the study and signed the Informed Consent.

2.2. Sample Collection A peripheral blood sample from each subject of the family S1074 enrolled in the study was collected by venipuncture in 5 mM EDTA tubes and genomic DNA was extracted using Chemagen MSM I (Magnetic Separation Module I, PerkinElmer, Massachusetts, MA, USA) according to the manufacturer’s instructions. DNA was quantified by the fluorometric method Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Massachusetts, MA, USA).

2.3. Targeted Next-Generation Sequencing The index case of the family (II:2) was subjected for the genetic screening for causative hearing-loss mutations by using a custom gene panel, OTO-NGS-v2, designed in our laboratory [20]. As the causative mutation was identified following this approach, whole exome sequencing (WES) on the individual II:2 was not further performed. OTO-NGS-v2 is based on IDT probes capture system that included 117 genes associated with NSSNHL. Sequencing of captured enriched-libraries was done on the Illumina MiSeq (Illumina, Inc., San Diego, CA, USA). The sequence data were mapped against the se- quence (build GRCh37/hg19), and data analysis was performed using the Sophia Genetics’ software that enables the single nucleotide variations (SNVs) and the copy number varia- tion (CNV) analysis of the targeted exonic sequences. Variant prioritization was carried out using a custom filtering strategy [20].

2.4. Sanger Sequencing The c.832_836delCCTCA mutation in exon 8 of HOMER2 [NM_199330.3, long transcript] was verified by Sanger sequencing (Figure 1B). Briefly, a forward and a reverse oligonucleotide were designed for amplification of exon 8 (F-oligo 50-CGTGCACACATTGGTGATTT-30 and R-oligo 50-AAGCAGGAAAATGAGTACCATGA-30) followed by Sanger sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s directions in an ABI 3730S sequencer (Perkin Elmer, Waltham, MA, USA). The specificity of the primers designed for the amplification and Sanger sequencing of exon 8 of HOMER2 was confirmed by BlastN (https://blast.ncbi.nlm. nih.gov/; access date: 9 March 2021). We obtained a unique blast hit for the amplimer at (NC_000015.10; coordinates 82,851,291 to 82,851,310) within the HOMER2 genomic region. Segregation analysis was performed by checking the presence of the c.832_836delCCTCA mutation in all the affected and unaffected family members.

3. Results 3.1. Clinical Description of the Family The clinical history and audiological assessments of the affected members reported a post-lingual bilateral progressive hearing loss consistent with an autosomal dominant inheritance pattern (Figure1A). Individuals II:2 (8 years old) and II:3 (15 years old) exhibited moderate hearing loss with greater impact on the high frequencies (downsloping profile). Their mother (patient I:2, 39 years old) showed a more severe phenotype, displaying profound hearing loss at frequencies higher than 2000 Hz. The father (I:1, 44 years old) and his healthy son (II:1, 11 years old) showed normal hearing thresholds (Figure1C) at the time of the study. Genes 2021, 12, x FOR PEER REVIEW 4 of 11

3. Results 3.1. Clinical Description of the Family The clinical history and audiological assessments of the affected members reported a post-lingual bilateral progressive hearing loss consistent with an autosomal dominant in- heritance pattern (Figure 1A). Individuals II:2 (8 years old) and II:3 (15 years old) exhibited moderate hearing loss with greater impact on the high frequencies (downsloping profile). Their mother (patient I:2, 39 years old) showed a more severe phenotype, displaying pro- found hearing loss at frequencies higher than 2000 Hz. The father (I:1, 44 years old) and Genes 2021, 12, 411 4 of 9 his healthy son (II:1, 11 years old) showed normal hearing thresholds (Figure 1C) at the time of the study.

FigureFigure 1. (A 1.) (PedigreeA) Pedigree of the of the S1074 S1074 family family indicating indicating the the segregation segregation of of c.832_836delCCTCA c.832_836delCCTCA mutationmutation in inHOMER2 HOMER2. Black. Black symbolssymbols indicate indicate affected affected patients patients (ca (carryingrrying the the mutation mutation in in heterozygosis), heterozygosis), and white symbolssymbols correspondcorrespond to to normal normal hearinghearing individuals individuals (wild-type (wild-type for for the the muta mutationtion studied). studied). The The subject subject pointed pointed with an arrow isis thethe index index case case (studied (studied by by OTO-NGS-v2OTO-NGS-v2 panel), panel), and and the the ones ones marked marked wi withth an an asterisk asterisk were were analysed analysed by by Sang Sangerer sequencing forfor segregationsegregation analysis. analysis. (B) (ElectropherogramsB) Electropherograms corresponding corresponding to tothe the wild-type wild-type (left) (left) and and mutant mutant (right) (right) sequences ofof aa normalnormal hearing hearing and and an an affectedaffected individual, individual, respectively. respectively. (C) ( CAudiograms) Audiograms of ofthe the S1074 S1074 family. family. The The data data represented represented correspond correspond to to the the average average of the audiometricof the audiometric threshol thresholdsds in both in ears. both ears.(D) Integrative (D) Integrative Genomics Genomics Viewer Viewer (IGV) (IGV) (Bro (Broadad Institute) Institute) screenshot screenshot showing showing the genomicthe genomic region corresponding region corresponding to the c.832_836delCCTCA to the c.832_836delCCTCA mutation mutation of HOMER2 of HOMER2 and theand translated the translated protein protein stretch stretch corre- sponding to exon 8 in the reverse orientation (NP_955362.1, HOMER2 long isoform, amino acid range 292–266), as corresponding to exon 8 in the reverse orientation (NP_955362.1, HOMER2 long isoform, amino acid range 292–266), as HOMER2 is transcribed by using the DNA negative strand. (E) Schematic representation of the structure of HOMER2 long HOMER2 is transcribed by using the DNA negative strand. (E) Schematic representation of the structure of HOMER2 long isoform (NP_955362.1). The different domains [7,14,21] and the mutations identified so far associated with hearing loss isoform (NP_955362.1). The different domains [7,14,21] and the mutations identified so far associated with hearing loss (in (in grey) are shown. The mutation identified in this work is shown in black. EVH1 (1–111 aa): Enabled/vasodilator-stimu- latedgrey) phosphoprotein are shown. The (Ena/VASP) mutation identified homology in this1; Coiled- work iscoil shown domain in black. (184–328 EVH1 aa); (1–111 CBD aa): (202–294 Enabled/vasodilator-stimulated aa): CDC42-binding do- main.phosphoprotein LZA (249–307 (Ena/VASP)aa): Leucine homology Zipper-A; 1; LZB Coiled-coil (322–350 domain aa): Leucine (184–328 Zipper-B. aa); CBD (202–294 aa): CDC42-binding domain. LZA (249–307 aa): Leucine Zipper-A; LZB (322–350 aa): Leucine Zipper-B.

3.2. Genetic Study By using the OTO-NGS-v2 panel and Sophia DMM software 338 heterozygous genetic variants were retained in the index case (II:2) of the family, 10 of which were classified as potentially pathogenic in HOMER2, MYO7A, TMPRSS3, COL11A2, GPSM2, BDP1, EPS8, EPS8L2, OSBPL2, and PCDH15 genes, respectively (Table1). During the tertiary analysis, 9 variants were discarded. Two of them showed high population frequencies in the Genome Aggregation Database (GnomAD) and were classified as benign variants in ClinVar (MYO7A, COL11A2). The other 6 variants were associated with recessive forms of deafness (TMPRSS3, GPSM2, BDP1, EPS8, EPS8L2, and PCDH15). The variant c.747A>G (rs1309059934) in OSBPL2 (DFNA67) was classified by Sophia DMM as poten- Genes 2021, 12, 411 5 of 9

tially pathogenic. However, it resulted in a synonymous protein change (p.Arg249Arg) that was classified as likely benign (BP4, BP7, and PM2) according to the American College of Medical Genetics and Genomics (ACMG) guidelines [22,23]. Furthermore, we confirmed by Sanger sequencing that this mutation did not segregate with the hearing loss in the family as it was detected in the normal-hearing subject II:I. Finally, a novel mutation in HOMER2 gene, c.832_836delCCTCA, was identified in the propositus of the S1074 family (patient II:2). This variant was identified at exon 8 of HOMER2 and is supposed to alter the two transcript variants with known reported expression (NM_004839.4, short transcript, and NM_199330.3, long transcript). The variant leads to a frameshift generating a premature stop codon that in the absence of nonsense- mediated decay (NMD) may produce a truncated HOMER2 protein (p.Pro278Alafs*10). This mutation was classified as pathogenic (PVS1, PM2 and PP3) according to the ACMG guidelines [22,23]. The variant was not present in the GnomAD [24], in the Collaborative Spanish Variant Server-CSVS database [25] nor in the Deafness Variation Database (DVD, http://deafnessvariationdatabase.org/; access date: 9 March 2021;) [26,27]. Additionally, we confirmed by Sanger sequencing (Figure1B) it segregated with the hearing loss in the family. The novel HOMER2 variant c.832_836delCCTCA has been deposited in ClinVar (accession# SCV001499845). Comparison of the genetic and clinical data of the S1074 family with previously reported cases is made in Table2.

4. Discussion Hearing loss caused by HOMER2 mutations is an extremely rare disorder. In this study, we have used OTO-NGS-v2, a custom targeted NGS panel, for the identification of a novel mutation (c.832_836delCCTCA; p.Pro278Alafs*10) in this gene linked to DFNA68 hearing loss. To date, only two different mutations in HOMER2 have been documented to cause hearing loss. The first mutation was a missense substitution (c.587G>C; p.Arg196Pro; NM_199330.3) that affects a highly conserved residue in the coiled-coil (CC) structure, a region that is required for homo/hetero-multimerization to form tetrameric hubs (in which the CC domains align in a parallel fashion) and for interaction through the CBD with Rho family GTPase proteins like CDC42, a GTPase that mediates -turnover [28] and is responsible for planar polarity establishment in hair cells [29,30]. Functional assessment of the p.Arg196Pro in zebrafish strongly suggests that this mutation exerts its effect through a dominant-negative mechanism on wild-type protein by either inhibiting multimerization or competing for partner proteins [18]. The second mutation, c.840_840dup, p.Met281Hisfs*9 (NM_199330.3) was identified in a Chinese family with symmetric ADSNHL [19]. This frameshift variant at the CDC42-binding domain leads to the generation of a premature stop codon supposed to produce a truncated HOMER2 protein of 288 amino acids, with an aberrant protein tail of 8 amino acids from position 281 onwards. In this work, we have identified a novel frameshift mutation, c.832_836delCCTCA, p.Pro278Alafs*10 in HOMER2 that represents the second truncating mutation identified in the CDC42-binding domain. The Spanish mutation also creates a premature stop codon that may lead to generate a truncated shorter protein of 286 amino acids with an aberrant tail of 9 amino acids from position 278, thus lacking the canonical C-terminal end. Interestingly, the alignment of both truncated proteins revealed an identical sequence of the last 8 amino acids of the aberrant tails (Figure2). Genes 2021, 12, 411 6 of 9

Table 1. Genetic variants with potential pathogenicity identified by SOPHIA DDM software in the index case (II:2) of family S1074.

Pathogenicity Gene c.DNA and Variant Fraction ClinVar Exon Coding Consequence Prediction by ACMG GnomAD Transcript Protein Alteration Coverage (Ref/Alt) Rating SOPHIA DDM HOMER2 c.832_836delCCTCA 34.98% Pathogenic 8 frameshift Highly Pathogenic - 0 DFNA68 NM_199330 p.Pro278Alafs*10 (145/78) (PVS1, PM2, PP3) MYO7A c.3515_3536del 49.0% Benign Benign DFNA11 27 No-stop Highly Pathogenic 0.391 NM_001127179 p.Gly1172*1179del (582/306) (PVS1, BA1, BS2, BP6) rs111033223 DFNB2 TMPRSS3 46.73% Splice Benign DFNB8 8 c.617-3_617-2dupTA p.? Highly Pathogenic (PVS1, PP3, BA1, BP6) 0.118 NM_001256317 (421/372) acceptor rs34966432 DFNB10 COL11A2 50.43% Potentially Benign Benign DFNA13 32 c.2307 + 3G>A p.? splice_donor_+3 0.601 NM_080679 (402/409) Pathogenic (BA1, BS2) rs970901 DFNB53 GPSM2 c.1572_1574delTTC 48.55% Potentially Benign 13 inframe_3 Benign(PP3, BA1, BP3, BP6) 0.291 DFNB82 NM_013296 p.Ser525del (278/267) Pathogenic rs35029887 BDP1 c.5068G>C 44.54% Potentially Likely Benign - 23 missense 7.48 × 10−5 DFNB112 NM_018429 p.Gly1690Arg (335/269) Pathogenic (PM2, BP1, BP4) rs193135814 EPS8 c.2230G>A 44.34% Potentially Benign Likely Benign 20 missense 2.06 × 10−3 DFNB102 NM_004447 p.Val744Ile (359/286) Pathogenic (BS1, BS2, BP1, BP6) rs77967764 EPS8L2 c.710G>T 50.22% Potentially VUS 9 missense - 7.1 × 10−6 DFNB106 NM_022772 p.Arg237Leu (560/565) Pathogenic (PM2, BP4) OSBPL2 c.747A>G 51.09% Potentially Likely Benign - 9 synonymous 0 DFNA67 NM_014835 p.Arg249Arg (157/164) Pathogenic (BP4, BP7, PM2) rs1309059934 Uncertain PCDH15 c.2596G>A 49.09% Potentially VUS 21 missense Significance 6.37 × 10−4 DFNB23 NM_001142771 p.Val866Met (446/430) Pathogenic (PM1, PM2) rs142512524

Table 2. Clinical data and classification of the different variants described in HOMER2 causing hearing loss.

Detection Audiogram Number of Scores Supporting CSVS GnomAD DNA Change Protein Change Exon Origin Phenotype Degree ACMG Classification DVD References Decade Profile Pathogenicity Pathogenicity Allele Freq Allele Freq. European Mild- Down- Uncertain significance Azaiez et al. c.587G>C p.Arg196Pro 6 SNHL 1st 20/22 N.A 0 0 descent profound sloping (PM2, PP3, PP5, BP1) 2015 Moderate- Down- Pathogenic c.840_840dup p.(Met281Hisfs*9) 8 Chinese SNHL 1st N.A N.A 0 0 Lu et al. 2018 profound sloping (PVS1, PM2, PP3, PP5) Moderate- Down- Pathogenic c.832_836delCCTCA p.(Pro278Alafs*10) 8 Spanish SNHL 1st N.A N.A 0 0 This work profound sloping (PVS1, PM2, PP3) All the variants are named according to NM_199330.3 long transcript and the nomenclature was checked using Mutalyzer 2.0.32. ACMG criteria [22,23]: PVS1 (Pathogenic Very Strong): null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where LOF is a known mechanism of disease. PM2 (Pathogenic Moderate 2): absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium. PP3 (Pathogenic Supporting 3): multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). PP5 (Pathogenic Supporting 5): reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation. BP1 (Benign Supporting 1): missense variant in a gene for which primarily truncating variants are known to cause disease. The databases GnomAD [24], CSVS [25], and Deafness Variation Database (DVD) [26] were searched on the 20th December 2020. SNHL: sensorineural hearing loss, NA: not available. Genes 2021, 12, 411 7 of 9 Genes 2021, 12, x FOR PEER REVIEW 8 of 11

Figure 2. Alignment of the wild-type protein fragment encoded by exon 8 of HOMER2 long isoform (NP_955362.1) and Figure 2. Alignment of the wild-type protein fragment encoded by exon 8 of HOMER2 long isoform (NP_955362.1) and the truncating mutations in the CDC42-binding domain (CBD) identified in the Chinese (p.Met281Hisfs*9) and Spanish the truncating mutations in the CDC42-binding domain (CBD) identified in the Chinese (p.Met281Hisfs*9) and Spanish (p.Pro278Alafs*10)(p.Pro278Alafs*10) families. families. The The amino amino acid acid sequence sequence shared shared between between both both aberrant aberrant protein protein tails tails is isshown shown in inbold bold face. face. Based on the similar effect that the two different frameshift mutations may cause Based on the similar effect that the two different frameshift mutations may cause at at the protein level, both supposed to truncate the protein at the CDB and resulting in the protein level, both supposed to truncate the protein at the CDB and resulting in the the same aberrant tail, it might be reasonable to suggest that the Chinese and Spanish same aberrant tail, it might be reasonable to suggest that the Chinese and Spanish muta- mutations may share a similar mechanism of pathogenesis. Lu et al. demonstrated that tions may share a similar mechanism of pathogenesis. Lu et al. demonstrated that the the p.Met281Hisfs*9 mutant protein was less stable than the wild-type protein and it p.Met281Hisfs*9showed an altered mutant subcellular protein was localization less stable in than HEK293T the wild-type and HEI-OC1 protein cells. and Whereasit showed the anwild-type altered subcellular proteins were localization mainly aggregatedin HEK293T near and the HEI-OC1 nucleus, cells. the Whereas p.Met281Hisfs*9 the wild-type mutants proteinswere morewere widelymainly distributedaggregated throughoutnear the nucleus, the cytoplasm. the p.Met281Hisfs*9 Furthermore, mutants these authorswere moredemonstrated widely distributed that p.Met281Hisfs*9 throughout the showed cytoplasm. a decreased Furthermore, ability these to oligomerize authors demon- [19]. It stratedhasalso that beenp.Met281Hisfs*9 postulated thatshowed HOMER2 a decreased could ability play an to importantoligomerize role [19]. in It maintaining has also beenstereocilia postulated through that HOMER2 its interaction could with play CDC42 an important [19], therefore role in the maintaining truncation ofstereocilia HOMER2 throughin the its CBD interaction could eventually with CDC42 prevent [19], therefore its interaction the truncation with other of HOMERHOMER2 protein in the CBD family couldmembers eventually and withprevent other its interaction proteins like with CDC42. other HOMER Indeed, protein targeted family deletion members of Cdc42 and in withmurine other hairproteins cells causeslike CDC42. a progressive Indeed, targeted hearing lossdeletion phenotype of Cdc42 that in is murine comparable hair cells to the causeshearing a progressive loss phenotype hearing in loss the phenotype Spanish and that Chinese is comparable families to [19 the]. Anotherhearing loss possibility phe- notypeis that in thethe HOMER2Spanish andmutation Chinese identifiedfamilies [19]. in thisAnother study possibility may cause is that nonsense-mediated the HOMER2 mutationdecay (NMD),identified a mechanismin this study thatmay affects cause nonsense-mediated the processing of the decay transcripts (NMD), ata mecha- different nismextent that dependingaffects the processing on the type of of the mutation transcripts and atgene different involved extent as depending previously on reported the type in of othermutation pathologies and gene like involved Neurofibromatosis as previously I reported [31]. In this in other regard, pathologies and in contrast like Neurofi- to mouse bromatosismutants homozygousI [31]. In this forregard, the targeted and in deletioncontrast ofto Homer2mouse mutantsthat display homozygous early-onset for rapidly the targetedprogressive deletion hearing of Homer2 loss, micethat display heterozygous early-onset for the rapidly targeted progressive deletion hearing of exon loss, 3 in Homer2 mice heterozygous(Homer−/+) displayedfor the targeted normal deletion hearing of levels exon [ 183 ].in ItHomer2 may indicate (Homer that−/+) displayed a moderate normal or even hearinglow extent levels NMD [18]. mightIt may be indicate associated that with a moderateHOMER2 orframeshift even low mutations extent NMD thus might suggesting be associatedthat the pathophysiologywith HOMER2 frameshift of the DFNA68 mutations hearing thus loss suggesting in the Spanish that the and pathophysiology Chinese families of wouldthe DFNA68 not be hearing mediated loss by in haploinsufficiency, the Spanish and Chinese but by a families gain-of-function would not of thebe mediated Ct aberrant bytail haploinsufficiency, or by a dominant-negative but by a gain-of-function mechanism as itof hasthe beenCt aberrant postulated tail or for by the a p.Arg196Prodominant- negativemissense mechanism mutation as [18 it]. has However, been postulated more experiments for the p.Arg196Pro to detect HOMER2 missensemutant mutation transcripts [18]. However,levels by more using experiments a gene-editing to detect tool to HOMER2 mimic actual mutant mutation transcripts in cell levels lines by or using the generation a gene- editingof knock-in tool to murine mimic modelsactual mutation are necessary in cell to fullylines understandor the generation the underlying of knock-in mechanism murine of modelspathogenesis are necessary linked to DFNA68fully understand frameshift the mutations. underlying mechanism of pathogenesis linked toRegarding DFNA68 theframeshift clinical mutations. phenotype, the hearing loss observed in HOMER2 patients causedRegarding by the the missense clinical (p.Arg196Pro) phenotype, the and hearing two truncating loss observed mutations in HOMER2 seems topatients be quite causedsimilar by in the the missense three studied (p.Arg196Pro) families. Affectedand two individualstruncating mutation show progressives seems to hearing be quite loss similaraffecting in the mainly three thestudied high families. frequencies Affected (downsloping individuals profile) show withprogressive a typical hearing onset loss in the affectingfirst decade mainly of the life high (7–9 frequencies years old), although(downsloping a more profile) severe with phenotype a typical was onset detected in the in firstpatients decade bearing of life the(7–9 truncating years old), mutations although when a more age-matched severe phenotype hearing-impaired was detected subjects in of patientsthe three bearing families the weretruncating compared. mutations For individuals when age-matched in a similar hearing-impaired age-range, those subjects carrying of the missensethree families p.Arg196Pro were compared. mutation For [18 indi] displayedviduals in minor a similar affectation age-range, on the those entire carrying range of thefrequencies missense p.Arg196Pro (i.e., subject mutation IV:2, 8y.o, [18] IV:10, disp 15y.o,layed and minor III:4, affectation 34y.o, European on the entire ascent range family) of thanfrequencies patients (i.e., carrying subject the IV:2 p.Met281Hisfs*9, 8y.o, IV:10, 15y.o, (subject and III:4, IV:7, 34y.o, 7y.o; III:10, European 34y.o, ascent and III:8, family) 39y.o thanin thepatients Chinese carrying family) the or p.Met2 the p.Pro278Alafs*1081Hisfs*9 (subject variant IV:7, (subject7y.o; III:10, II:2, 34y.o, 8y.o; II:3,and 15y.o,III:8, 39y.o and I:2, in 39y.othe Chinese in the Spanish family) family).or the p.Pro278Alafs*10 The presence of tinnitus variantor (subject cranial II:2, tinnitus, 8y.o; however,II:3, 15y.o, has and only I:2,been 39y.o displayed in the Spanish by some family). affected The members presence ofof thetinnitus Chinese or cranial family tinnitus, and this however, phenotype has was only been displayed by some affected members of the Chinese family and this phenotype was not present in any of the other two families. The identification of other HOMER2 cases

Genes 2021, 12, 411 8 of 9

not present in any of the other two families. The identification of other HOMER2 cases is, therefore, necessary to further establish more accurate genotype–phenotype correlations. In summary, we have identified a third variant in HOMER2; the first one reported in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations. Our study also highlights the importance of using NGS-based diagnostic methods to identify mutations in low-prevalence deafness genes like HOMER2, thus helping to improve our knowledge about the pathophysiology of DFNA68 and to define more accurate genotype–phenotype correlations in this disorder.

Author Contributions: Conceptualization, M.M. and M.Á.M.-P.; Data curation, M.L., M.V. and I.d.C.; Formal analysis, M.L. and M.M.; Funding acquisition, M.Á.M.-P.; Methodology, M.M.; Supervision, M.Á.M.-P.; Writing—original draft, M.L., M.M. and M.Á.M.-P. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by grants from the Spanish Institute of Health Carlos III (ISCIII) cofunded with the European Regional Development Fund (ERDF) within the “Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020” (PI14-948, PI17-1659 and PI20/0429 to MAMP), the Spanish Center for Biomedical Network Research on Rare Diseases (CIBERER, 06/07/0036 grant, to MAMP) and by the Regional Government of Madrid (CAM, B2017/ BMD3721 grant to MAMP). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Hospital Ramón y Cajal (protocol code 288-17; 29 January 2018). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are openly available in ClinVar (accession# SCV001499845). Acknowledgments: The authors sincerely thank the family for their participation in this study. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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